Sensitive Analysis of Antiepileptic Drugs in Very Small Portions of Human Brain by Microbore HPLC

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A method for the simultaneous determination of phenobarbital (PB), phenytoin (PT), carbamazepine (CBZ), carbamazepine-10,11- epoxide (EPO), and N-desmethyl methsuximide (DM) in very small amounts of human brain (20–100 mg) is described. The weighed tissue is homogenized with a neutral buffer solution containing the internal standard (IS) 5-ethyl-5-(p-tolyl)-barbituric acid (ETB). The antiepileptic drugs (AEDs) are extracted with acetone from the homogenate. After centrifugation the drug solution is transferred onto an Extrelut(R)-III tube (filled with kieselguhr) and an acidic buffer is added. The AEDs are extracted from the matrix by di-chloro methane/propanol-(2) (97:3). The solvents are evaporated off and the residues taken up in methanol/water. The concentrated drug solution is injected into a microbore column which is filled with ODS silica gel (5 um). The mobile phase for the gradient elution is a mixture of a 0.05 M ammonium di-hydrogen phosphate buffer (pH 4.4) and acetonitrile. The column temperature is 65°C and the detection wavelength 207 nm.